The invention herein relates to the field of immuno-chemical assays of pesticides. In particular the specific method described and claimed relates to an enzyme-linked immunosorbent assay ("ELISA") for the detection of .alpha.-haloacetamides.
Immunoassays are rapidly becoming an important technique in the analysis of pesticide residues. As used herein the term "pesticides" refer to chemicals used to control weeds or animal pests in agricultural crops.
Basic immunoassay techniques in use today for detecting pesticides or analytes, include those called the "sandwich", "labeled analyte" and "second antibody", e.g., enzyme-linked immunosorbent assay ("ELISA") methods. A modification of the labeled analyte method is known as an enzyme multiplied immuno technique ("EMIT"). The methods will be described in more detail below.
In any immunoassay, the antibodies are the essential reagent which provides the specificity and dictate the ultimate level of sensitivity which can be achieved. Antibodies are immunoglobulin protein molecules produced as part of the mammalian immunodefense system. Gamma-immunoglobulins (IgG), which are the antibody proteins most frequently used in immunoassays, have molecular weights of about 160,000 and consist of two variable binding regions per molecule and a non-binding region with a constant amino acid sequence which is characteristic for each animal species. These proteins are normally found in the blood and lymph systems of mammals and are commonly obtained from a blood sample by removal of the blood cells by clotting or centrifugation. The resulting preparation is the serum, which is often called "antiserum" or "immune serum" when it contains antibodies of interest. Such an antiserum contains a mixed population of many antibodies, some of which are directed toward the analyte of interest but most of which have resulted from other foreign substances to which the animal has been exposed. Because of this heterogeneous nature, antisera are also often called polyclonal antibodies.
Nearly all of the pesticide immunoassays described in the literature have employed rabbit antisera as their source of antibodies, at least in the initial stages. Since the population of antibodies in an animal's blood can vary over time, a single sample obtained from one bleeding or by pooling several bleedings is usually used throughout the experimental period. Other animals such as mice, goats or horses can be used to supply antibodies, but rabbits are particularly easy to keep and work with. Most investigators have simply used diluted antisera in their assays, although some have purified the immunoglobulins by precipitation or immunoaffinity columns. The major advantages of using purified antibodies are realized if there is an unwanted antibody population in the antiserum which leads to unacceptable interferences, although increased sensitivity can also be realized in some assay formats. If the investigator wants to covalently link a label or tracer to the antibody for use in a sandwich assay as described below it is also essential to purify the antibody first. Using modern methodology, antisera can often be diluted 1:1000 or more with buffer prior to their use in immunoassays, allowing many assays to be completed with a single blood sample.
In some studies in the literature, monoclonal antibodies are employed. These antibodies are obtained from the medium fluids of hybridoma cell lines or from ascites tumors produced in mice intentionally immunized with an appropriate hybridoma. The hybridoma cell lines are produced by a lengthy procedure initiated from spleen cells of mice which have been previously induced to produce the desired antibodies in the same way as the rabbits. The sequence of operations required to produce and identify useful monoclonal antibodies must be performed by someone with prior experience, since many specialized tasks and equipment are necessary. Such a process is best reserved for those cases where promising antisera have already undergone preliminary study and a clear intention to utilize the resulting hybridomas has been established. Such monoclonal antibodies are advantageous because a single homogeneous immunoglobulin is produced which may have a very narrow, well-defined specificity, which will be invariant from batch to batch, and which can be made available in large quantities. These characteristics make monoclonal antibodies particularly attractive for the production of diagnostic kits.
Because of their low molecular weight, there is a problem in the production of antibodies directed against pesticides. Unlike larger molecules, low molecular weight pesticide analytes, must be conjugated to a carrier protein, prior to immunizing the animals, because the free pesticide itself is too small to elicit antibody production even though it can bind to the antibodies once they are formed. This trait defines the pesticide as a "hapten" in immunological terms. The pesticide moiety should be covalently attached to the carrier; usually proteins such as bovine serum albumin (BSA), ovalbumin (OA), human serum albumin (HSA), or keyhole limpet hemocyanin (KLH) are used as carriers. The extent to which the hapten, after conjugation to the protein, resembles the free pesticide spatially and electronically will influence the specificity and sensitivity of the assay. The choice of protein carrier may be influenced by the projected use of the assay; for example, HSA should not be used if the assay will be used for analysis of human serum samples.
A careful strategy must be developed to design the hapten conjugate to achieve the desired assay specificity. In general, the portions of the hapten molecule which are more distant from the point of attachment to the protein carrier will have more influence on the antibody specificity than those used to accomplish the linkage. Formation of an amide bond to pendant lysine amino groups on the protein using carbodiimide reagents or other types of carboxyl activation have most commonly been used to attach the hapten, although nearly any kind of stable covalent linkage can be used. Often analogs or metabolites of the target pesticide with chemical functionality amenable to linkage formation are very useful. In addition, a spacer molecule which is commonly two to six carbon atoms long may be incorporated between the hapten and the carrier. Some investigators carefully determine the number of haptens attached to each molecule of carrier, which can best be determined using radiolabeled haptens or less accurately by UV absorbance, but others simply immunize the animals to determine if the conjugation reaction was successful. Although there is no clear agreement as to the ideal number of haptens per molecule of carrier, many investigators seem to target a ratio of one hapten for each 5000 to 20,000 daltons of protein molecular weight. As the prior discussion suggests, there are no rules for conjugate preparation, and several possible approaches should be considered or even attempted simultaneously. It is often useful to prepare conjugates using more than one carrier since subsequent steps will require the use of a second hapten conjugate to eliminate responses from antibodies directed toward the protein carrier rather than the hapten; a positive reaction with different proteins bearing the same hapten is good preliminary evidence that the antibodies may have desirable properties. In addition, if it becomes worthwhile to purify the antibodies using affinity columns, a second conjugate will be useful.
Animals are usually immunized subcutaneously at several sites initially with about one milligram of the hapten conjugate ("immunogen" or "antigen") per kilogram of body weight in Freund's complete adjuvant. This preparation contains heat-killed bacteria which enhance the animal's immune response. Subsequent booster immunizations are usually made on a regular schedule using about 20% of the original immunogen in Freund's incomplete adjuvant. Blood is then withdrawn from the animals at a fixed period of time, such as ten days after each boost, commencing four to eight weeks after the initial injection.
A second type of antibody is sometimes mentioned in the literature as part of the assay strategy. As discussed below, most assay techniques require a step in which the analyte-antibody complex must be physically separated from the rest of the mixture. One convenient approach is to use a second antibody from a different animal species which has been developed against the invariant part of the original (for example, rabbit) immunoglobulin. Such "second antibodies" are commonly called "goat-anti-rabbit" or GAR, for example, and are commercially available from immunological suppliers, often with convenient tracer molecules already covalently bound to them.
Once the antibodies have been obtained from the animal source, they must be evaluated for applicability toward analysis of the pesticide of interest. The formation of a complex through non-covalent binding of the antibody to the pesticide, much like the binding of an enzyme to its substrate, is of prime importance in the immunoasssay. All other phases of the process are designed to detect and quantitate the extent of formation of this complex. A variety of approaches has been devised toward this end.
In the early 1980's, solid phase immunoassay techniques were developed and have replaced older methods in nearly all assay protocols. Accordingly, the discussion below will be with reference to these solid phase techniques only.
Solid phase techniques rely on the adsorption of protein-hapten conjugates onto polystyrene or latex surfaces at high pH's. This non-covalent binding is essentially irreversible during the assay, and serves to immobilize a chosen protein without altering its immunological interactions. Although many geometric possibilities exist, by far most laboratory assays are performed in 96-well polystyrene "microtiter" plates which are available from many suppliers. After the desired conjugate is bound or "coated" onto the surface of each small well, the remaining binding sites are blocked using an inert protein such as gelatin or BSA. Although the binding capabilities of different plates vary, the availability of 96 wells on each plate combined with many types of automated liquid dispensing equipment allows the inclusion of standards in each analysis plate to overcome this problem. The immobilized conjugate can then be exposed to a series of reagent solutions, which are discarded after each step, separating those molecules which bind to the immobilized protein from all the rest. In addition, the capacity of each well is 200-300 microliters, so that the assay has been miniaturized as well as simplified. These improvements combined with the standardization of microtiter plate geometry has allowed the manufacture of equipment to process all 96 wells in such a plate simultaneously. With these features, quadruplicate analyses, for example, of many tens of samples can be done quickly using less than one milliliter of each sample.
There are three basic strategies used in modern immunoassays, only two of which appear applicable to pesticide analysis. These can be referred to as "sandwich", "labeled-analyte", and "second-antibody" methods. An essential part of each is the generation of calibration curves using known amounts of the desired analyte. The "sandwich" approach requires two antibodies which both recognize the analyte; both may actually be the same protein, one sample of which has been linked to a tracer molecule. The sandwich approach is attractive because the signal which develops in the sample wells at the assay's completion is directly proportional to the amount of analyte present and it does not rely on a competitive binding reaction. Unfortunately, it is not viewed as spatially possible to have two antibody molecules bound to a single small pesticide molecule simultaneously; thus, the sandwich technique is not applicable to pesticide analysis.
The "labeled-analyte" method is a conceptually simpler method than the second-antibody approach, and it is almost always used with radioimmunoassays (RIA). It requires that a sample of the pesticide be radiolabeled or covalently attached to an enzyme or fluorescent tracer. A constant known amount of this labeled pesticide is allowed to compete with the free pesticide in the unknown sample for the limited number of antibody binding sites attached to the well's surface. After washing away the unbound pesticide, both labeled and unlabeled, the amount of label remaining in the well is inversely proportional to the amount of pesticide originally in the unknown sample. In principle, the labeled analyte can have a slightly different structure than the measured analyte as long as the two molecules compete for the same binding site and the presence of the actual analyte inhibits the binding of the label in the concentration range of interest. This strategy has been used in the case of PCB's where many similar structures are of simultaneous analytical interest. The three most common types of labeled analyte involve radioactivity or the preparation of covalent conjugates of the analyte with fluorescent tags or enzymes.
The "second-antibody" method is commonly used in enzyme assays (EIA) such as the enzyme-linked immunosorbent assay (ELISA), because the required second antibodies, covalently labeled with an enzyme, are commercially available. In this approach, a protein conjugate of the pesticide using a different protein than that employed as the original immunogen, is coated onto the well's surface (called the "coating" or "screening" antigen). The fixed amount of hapten moieties on this coating antigen then compete with the free pesticide molecules in the unknown sample for the limited number of antibody binding sites. The interaction between the antibody and pesticide in the fluid phase inhibits the ability of the antibody to bind to the solid phase coating or screening antigen. Hence, when high concentrations of pesticide are present in the test sample, small concentrations of antibody will react with the solid phase coating antigen. Conversely, high concentration of antibodies will be bound to the solid phase when low levels of pesticides are present in the test sample. The antibodies bound to the solid phase are then detected by formation of another complex using a commercially available labeled second antibody directed toward the heavy chain constant portion of the pesticide-specific antibody. After washing out the unbound labeled second antibody, the label remaining in each well is inversely proportional to the amount of pesticide in the unknown sample.
In all immunoassay techniques, a final measurement must be made which can be correlated using a calibration curve to the amount of pesticide present in the unknown sample. The type of measurement will depend upon the nature of the label which was attached to the hapten, antibody, or second antibody in the assay strategy. Radioimmunoassay techniques were the most common assays in use a few years ago, and are still in wide use today. Although .sup.14 C-labeled samples of pesticides are usually available, the specific activities of these compounds cannot often be made high enough to allow accurate detection of picogram to nanogram amounts of pesticide. Since most pesticide immunoassays are competitive binding assays, the detection limit of the assay will depend directly upon the mass of labeled compound which is competing with the unknown sample, and therefore on the specific activity of the radiolabel. For this reason, tritium or .sup.125 I radiolabels are required to produce very sensitive immunoassays. The synthesis, purification, use, and disposal of such high specific activity radiolabels may often be a major hurdle standing in the way of utilization of radioimmunoassays for pesticide analysis in many laboratories.
An attractive alternative is the use of enzyme-labeled components in the immunoassay strategy. The quantitative measurement of the amount of enzyme present at the final stage of the assay is based upon the addition of an excess of a substrate which the enzyme can convert to an easily quantifiable product. Most often this has involved formation of a colored product via the action of the enzyme on a substrate without these properties. Typical examples include alkaline phosphatase/p-nitrophenyl phosphate or horseradish peroxidase/o-phenylenediamine combinations, although nearly any rapid enzyme/substrate reaction which produces a stable, easily quantified product could be used. The above two enzymes are commonly employed because they have high activity and can be covalently bound to a variety of "second antibodies" by simple chemical techniques. Galactose conjugates of fluorescent phenols such as .beta.-napthol or 4-methylumbelliferone can also be used in conjunction with a .beta.-galactosidase label to generate a fluorescent signal which is proportional to the enzyme concentration. In general, the colored products produced by horseradish peroxidase or alkaline phosphatase seem to be most conveniently measured by common laboratory equipment unless the sample matrix generates a competing signal. Certainly for use in field test kits, a visible signal which does not require complex equipment to detect a positive sample is preferable. In some cases, an additional non-covalent binding step has been used with enzyme labels which involves the tight binding between biotin (vitamin H) and avidin, a biotin-binding protein from egg whites. In this strategy, for example, biotin-labeled second antibodies are mixed with avidin-enzyme conjugates, usually to achieve an additional amplification of the ultimate enzyme signal. This strategy has not been specifically reported for pesticide analysis.
The third type of label which is sometimes used is a fluorescent label, which is distinguished from an enzyme label acting on a fluorescent substrate. In this case, the hapten or second antibody is covalently linked directly to the fluorophore, for example fluorescein. The fluorescent label can then function just like a radiolabel except that the final measurement involves the excitation and emission process. The fluorescent approach offers the potential advantage of increased sensitivity or diminished background signal when interferences are a problem without the need of radioactivity. The equipment for measuring fluorescence is not quite as commonly available as those for colorimetric determinations using enzyme labels. One adaptation which has been advocated in some pesticide analyses, apparently mainly as a proprietary foothold for kit development, is the fluorescence polarization technique. In this method, the optical rotation of the fluorescent emission from a polarized excitation beam is correlated to the degree of antibody-pesticide complex formed. It has not seen wide usage.
There is one other approach which is very attractive for pesticide immunoassays which involves a proprietary strategy developed by one company for application in clinical diagnostics. The method is called EMIT (Enzyme Multiplied Immuno Technique) and is a modification of the labeled-analyte approach. The analyte must be covalently conjugated to an enzyme label close to the active site of the enzyme so that complexing to the antibody spatially inhibits the enzyme reaction. When this hapten-enzyme conjugate is incubated with the test sample and the antibody in a homogeneous solution, no physical separation of the bound and unbound moieties is required because only those haptenenzyme conjugates which are unbound can be detected when the substrate is added. Therefore, only those samples which contain the pesticide will develop a detectable enzyme product since the presence of the free pesticide is required to prevent the inhibitory binding of the antibody to the enzyme-hapten conjugate.
Immunoassays inherently offer an extremely sensitive technique in relation to the amount of effort which must be expended. In most of the pesticide immunoassays, small aliquots of water, urine, serum, or extract samples can be used directly in the assay without further clean-up or concentration. This is in obvious contrast to many other instrumental methods where often tens or even hundreds of milliliters of a sample may have to be extracted, fractionated, and concentrated prior to the final analytical step. Using the direct immunoassay analysis format, the typical sensitivities reported for pesticide immunoassays have detection limits in the range 1-10 nanograms per milliliter (PPB) give or take one order of magnitude. These sensitivities are usually more than adequate for most pesticide analyses; obviously these levels could be lowered even further by applying the extraction and concentration procedures which have likely already been developed for most instrumental pesticide methods prior to the actual immunoassay step, although much of the potential time savings would then be sacrificed.
The working dynamic range for most immunoassays seems to span about two orders of magnitude, such as 1-100 parts per billion (PPB). When analysis of samples above the appropriate concentration range is attempted, most of the binding sites are already occupied, and the change in signal produced by the additional amounts of pesticide is undetectable. In many cases, the optimized immunoassay may actually be too sensitive for convenient direct assay of some pesticide samples, requiring that they be diluted prior to analysis or that the assay protocol be modified by increasing the number of total binding sites available, effectively reducing overall sensitivity. These problems are the direct result of the competitive binding nature of the assay format, and are inherent in the immunoassay strategy. For situations in which samples are expected to contain pesticide at concentrations spanning several orders of magnitude, a tiered approach may be useful in which several immunoassays are used to "sort" the samples into the most appropriate assay to achieve optimum precision. This can be accomplished readily because immunoassays lend themselves to automation very well so that doubling or even increasing the sample load by a factor of five will not overtax the system once all of the other details are worked out.
In general, immunoassays are very selective for the analyte of interest, which is the basis for their direct application to crude unpurified samples. Often this selectivity is evaluated in the literature by reference to a table of cross reactivities. This concept compares the intensity of the signal produced in the assay by a fixed amount of each of a group of compounds which might conceivably interfere. The amount is usually the amount of the desired analyte which produced about a 50% response. The intensity of the signal produced by the other compounds as compared to that of the desired analyte can then be expressed as "% cross reactivity". Alternatively, the same concept can be expressed by citing the concentration of each compound required to produce a 50% response in the assay, called ICso. The extent of cross reactivities of analogous compounds cannot be predicted. Immunoassays are capable of distinguishing one optical enantiomer from the other if such isomerism exists. In some cases, the similarity of other commercial compounds can present a substantial problem to use of the immunoassay, while in other cases the response of other analogs can be beneficial, particularly if analysis of metabolites or a group of similar compounds is desired. Generally, purification of polyclonal antibodies via affinity columns can be used to remove undesirable cross-reacting populations; the ultimate step in this approach is the use of a single monoclonal antibody which has been selected to have exactly the desired specificity.
Although a substantial number of immunoassays have been reported in the literature for pesticides and environmental contaminants, only those which can detect parathion, paraoxon, or paraquat in human serum as a result of poisoning appear to have been actually used extensively. A number of reasons can be suggested. Most of the compounds tested have not enjoyed great commercial success during the period for which the immunoassay has been available, suggesting that the desire for great numbers of analyses may not have materialized. The environmental contaminants such as DDT, PCB's, kepone, etc. have traditionally been measured using gas chromatography, which allows simultaneous detection of most of the compounds of concern in a single analysis. In this respect, the selectivity of an immunoassay is actually a disadvantage, and should be considered as one criterion in the decision regarding choice of potential analytical methodology. Immunoassays are advantageous from a time and sample-size standpoint, and the impact of these advantages multiplies as the number of required assays increases. The time required to develop an immunoassay to a usable stage is clearly greater than to develop a comparable instrumental method. Even though an immunoassay method may have been published, establishing it as a useful technique in another laboratory will require a substantial time investment. In addition, before the advent of enzyme (ELISA) or other kinds of labels, the requirement for use of radioisotopes almost certainly limited the utility of immunoassays.
Of the various other pesticides which have been analyzed by immunological methods, there are mentioned atrazine, chlorosulfuron, cyanazine, 2,4-D, diclofop-methyl, pentachlorophenol, 2,4,5-T and terbutryn. To our knowledge no immunoassay for .alpha.-haloacetamide herbicides has been described in the literature prior to this invention. And to our knowledge, only one literature reference even mentioned any .alpha.-haloacetanilides (viz. alachlor, metolachlor and propachlor) as cross-interferants in an immunoassay system, i.e., one designed to detect the fungicide metalaxyl. That system is described in an article by W. H. Newsome, J. Agric. Food Chem. 1985, 33, 528-530.
Accordingly, it is an object of this invention to provide an ELISA immunoassay system which provides novel antigens for raising novel antibodies in novel antisera for detecting .alpha.-haloacetamides, particularly .alpha.-chloroacetanilides, best exemplified by the commercial products alachlor, allidochlor, amidochlor, butachlor, metazalachlor, metolachlor, pretilachlor and propachlor.